Methods of obtaining rough strains of bacteria, such as those from the genus Mycobacterium, that are known in the art include the plating out of bacteria, such as those from the genus Mycobacterium, and looking for any rough colonies. However, any rough strains identified in this way are typically not stable.
Other methods include plating out Mycobacteria on medium that contains Tween or glycerol and encouraging pellicle growth. This method has been successful in inducing rough colonies of some species of Mycobacteria. However, this method has not been able to induce rough variants in all Mycobacteria. 
No method to date has been able to induce rough strains of Mycobacterium obuense. 
Accordingly, there is a need for an improved method that is able to produce rough strains, particularly stable rough strains, of Mycobacterium. Such a method could advantageously be used to produce for the first time rough strains of some species of Mycobacterium such as Mycobacterium obuense. 
Advantageously, the present invention has provided such an improved method that has induced for the first time a rough strain, particularly a stable rough strain, of Mycobacterium obuense. 
Rough strains of fast-growing Mycobacteria have a number of advantages:                stable rough strains do not revert to smooth strains;        rough strains have a different presentation of antigens and tend to be less virulent in causing disease than smooth strains;        skin test reagents made from rough strains are more effective than those made from smooth strains;        rough strains are very effective when used in in vitro cell-mediated immune experiments; and        the small clumps or particles of several bacilli may be more effective in stimulating phagocytic cells than single organisms of smooth strains.        
Furthermore, the present invention has advantageously found that a whole cell of a rough strain of Mycobacterium obuense may be used to modulate the immune response of a subject.
Although M. vaccae was known to modulate the immune response of a subject—M. obuense had never before been considered suitable for use as an immune modulator.
Vaccines and other immune modulators have a major impact in reducing morbidity and mortality from disease. The primary immunity elicited by most current vaccines appears to be mediated by the humoral immune response. For diseases that may require a cellular immune response, such as tuberculosis and leishmaniasis, there are currently no available vaccines that are uniformly effective.
Typically, adjuvants are added to vaccines. The role of the adjuvant is to enhance the body's immune response to specific antigens of the vaccine. Commonly used adjuvants typically produce a humoral immune response but not a cell-mediated immune response. In addition, aluminium adjuvants for example may cause negative side effects, such as sterile abscesses, erythema, swelling, subcutaneous nodules, granulomatous inflammation and contact hypersensitivity.
A vaccine or other immune modulator is sought that modifies a cellular immune response and in particular the T helper cell response, for example, the T helper cell 1 (Th1) and T helper cell 2 (Th2) response.
There are many different autoimmune diseases, and they can each affect the body in different ways. Many of the autoimmune diseases are rare. As a group, however, autoimmune diseases afflict millions of people.
Some autoimmune diseases are known to begin or worsen with certain triggers such as viral, parasitic and chronic bacterial infections. Other less understood influences affect the immune system and the course of autoimmune diseases include ageing, chronic stress, hormones and pregnancy.
Autoimmune diseases are often chronic, requiring lifelong care and monitoring, even when the person may look or feel well. Currently, few autoimmune diseases can be cured or made to go into remission with treatment.
Physicians most often help patients manage the consequences of inflammation caused by the autoimmune disease. In some people, a limited number of immuno-suppressive medications may result in disease remission. However, even if their disease goes into remission, patients are rarely able to discontinue medication. The long-term side effects of immunosuppressive medication can be substantial.
Initiation and progression of vascular injury is a complex, multi-factorial process, but there is growing evidence that inflammatory responses play a key role. Vascular injury is involved in the development of atherosclerosis, and in thrombotic processes that lead to acute ischaemic syndromes such as myocardial infarction, stroke and peripheral artery occlusion.
Immune mechanisms may be important in the development and maintenance of atherosclerosis and myointimal hyperplasia (MIH).
Myointimal Hyperplasia (MIH) can be considered as an exaggerated healing response to injury such as balloon angioplasty. A cascade of events results in: loss of the basement membrane, migration of vascular smooth muscle cells (VSMC) from the media into the intima, VSMC proliferation and phenotypic change to a more secretory fibroblastic cell type and increased production of extracellular matrix, which eventually leads to stenosis or occlusion of the vessel. It occurs after bypass grafting and balloon angioplasty and affects approximately 30% of such cases in clinical practice. It is the major cause of failure of such procedures and treatment of the resulting stenosed and blocked vessels/grafts is problematic. The underlying cellular mechanisms leading to MIH are not well understood and to date no therapy had been developed which can effectively prevent it. The clinical relevance of the current patent relates to the very large numbers of coronary artery angioplasties which are performed annually in the UK and world-wide. Although drug eluting stents are currently producing promising results they are unlikely to prevent restenosis completely. Any safe, relatively inexpensive adjunctive therapy, such as the immunotherapy proposed in this patent, would have a major clinical impact.
The mechanisms involved in immunotherapy against restenosis are complex and not completely elucidated. The endothelial injury caused by angioplasty may be exacerbated by the host immune response to hsp's. Hsp's are proteins produced by stressed cells which have been implicated in the pathogenesis and the pathophysiology of various immunological disorders including atherosclerosis (Xu Q et al. Arterioscler Thromb 1992; 12: 789-799). It is likely that they will be present on endothelial and smooth muscle cells in the region of an angioplasty. In effect the hsp acts as an autoantigen which can then be attacked by the immune system. This situation can be induced experimentally by immunising with a cross-reactive mycobacterial hsp (hsp65) which leads to endothelial damage in rabbits and mice (Xu Q, et al. Arterioscler Thromb 1992; 12: 789-799 and George J, et al. Circ. Res. 2000; 86: 1203-1210). The effect appears to be dependent on IL-4 secreted by Th2 lymphocytes, and is probably mediated by antibody George J, et al. Circ. Res. 2000; 86: 1203-1210 and Schett G, et al. J. Clin. Invest. 1995; 96: 2569-2577). The relevance of these observations to man is suggested by the ability of affinity-purified human antibody eluted from hsp65 columns to damage stressed human endothelial cells in vitro. This finding suggests that the antibody cross-reacts with hsp60 which is the human homologue of hsp65, and may be accessible to antibody when expressed on the membranes of stressed endothelial cells. It has been suggested that such antibodies binding to stressed endothelial cells may be a factor in producing coronary artery disease after heart transplantation (Crisp S J et al. J Heart Lung Transplant 1994; 81-91). Mukherjee et al (Thromb Haemost 1996; 75: 258-60) showed no association between preoperative antibody levels to hsp65 and coronary restenosis, but did show that those patients where levels of such antibodies dropped after angioplasty were less likely to restenose. In fact the role of antibodies to hsp could be complex, because patients with vascular disease have not only raised antibody, but also raised levels of the hsp themselves (Wright B H, et al Heart Vessels 2000; 15: 18-22). Thus an apparent fall in antibody levels may merely reflect an increase in levels of the protein. Moreover the hsp have regulatory effects, and bind to arterial smooth muscle cells, leading to enhanced survival without a requirement for internalisation (Johnson A D et al. Atherosclerosis 1990; 84: 111-119).
WO2004/022093 and UK application number 0404102.6 (both of which references are incorporated herein by reference) disclose an immune modulator composition or a pharmaceutical composition comprising a whole cell of a bacterium from the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukanmurella and Nocardioides. 
In addition, U.S. application Ser. No. 10/893,524 (incorporated herein by reference) discloses the use of a whole cell of a bacterium from the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides in a manufacture of a medicament for the treatment or prevention of post-weaning multisystemic wasting syndrome (PMWS) and/or porcine dermatitis and nephropathy syndrome (PDNS).
However, none of these documents teaches or suggests the use of whole cells of rough strain, preferably a stable rough strain, of M. obuense to modulate a cellular immune response.